DE69422304T3 - CYTOKINE ANTAGONISTS - Google Patents

Abstract

Heterodimer proteins comprising a soluble alpha specificity determining cytokine receptor component and the extracellular domain of a beta receptor component function as CNTF and IL-6 antagonsists.

Description

BACKGROUND OF THE INVENTION

Even though they have been discovered due to various biological activities include ciliary neurotrophic factor (CNTF), leukemic inhibitory factor (LIF), Oncostatin M (OSM) and interleukin-6 (IL-6) a newly defined Family of cytokines (referred to herein as the "CNTF family" of cytokines). These cytokines are due to their distant structural similarities grouped together [Bazan, J., Neuron 7: 197-208 (1991); Rose and Bruce, Proc. Natl. Acad. Sci. USA 88: 8641-8645 (1991)] and, perhaps more importantly, because they share common "β" signal transduction Receptor components have [Baumann, et al., J. Biol. Chem. 265: 19853-19862 (1993); Davis, et al., Science 260: 1805-1808 (1993); Gearing, et al., Science 255: 1434-1437 (1992); lp, et al., Cell 69: 1121-1132 (1992); Stahl, et al., J. Biol. Chem. 268: 7628-7631 (1993); Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. receptor activation through this family of cytokines follows either homo- or heterodimerization of these β-components [Davis, et al., Science 260: 1805-1808 (1993); Murakami, et al., Science 260: 1808-1810 (1993); Steel and Yancopoulos, Cell 74: 587-590 (1993)]. IL-6 receptor activation requires homodimerization of gp130 [Murakami, et al., Science 260: 1808-1810 (1993); Hibi, et al. Cell 63: 1149-1157 (1990)], a protein originally when the IL-6 signal transducing molecule was identified [Hibi, et al., Cell 63: 1149-1157 (1990)]. CNTF, LIF and OSM receptor activation follows from heterodimerization between gp130 and a second gp130-related protein known as LIFRβ [Davis, et al., Science 260: 1805-1808 (1993)], originally due to his ability LIF binding [Gearing, et al., EMBO J. 10: 2839-2848 (1991)].

In addition to the β-components Some of these cytokines also require specificity determining "α" components, the are more limited in their tissue distribution than the β components and in this way the cellular targets of each cytokine [Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. So are LIF and OSM includes acting factors that may just be the present from gp130 and LIFRβ require the responsive cells while CNTF requires CNTFRα [Stahl and Yancopoulos, Cell 74: 587-590 (1993)], and IL-6 IL-6Rα requires [Kishimoto, et al., Science 258: 593-597 (1992)]. Both CNTFRα [Davis, et al., Science 259: 1736-1739 (1993)] as well as IL-6Rα [Hibi, et al., Cell 63: 1149-1157 (1990); Murakami, et al., Science 260: 1808-1810 (1990); Taga, et al., Cell 58: 573-581 (1989)] their function as soluble Exercise proteins, in accordance with the idea that they do not interact with intracellular signaling molecules, but that they serve to help their ligands with the suitable signal transducing β-subunits to interact [Stahl and Yancopoulos, Cell 74: 587-590 (1993)].

additional Evidence from other cytokine systems also supports the notion that Dimerization represents a general mechanism by which all cytokine receptors initiate signal transduction. growth hormone (GH) is perhaps the best example in this regard. crystallographic Investigations have revealed that every GH molecule contains two separate receptor binding sites, both of which binding domain Recognized in the receptor, in this way it is a single molecule GH possible is, two receptor molecules to bind [de Vos, et al., Science 255: 306-312 (1992)]. The dimerization takes place afterwards, wherein the binding site 1 on the GH first binds to a receptor molecule, followed by binding of binding site 2 to a second receptor molecule [Fuh, et al., Science 256: 1677-1680 (1992)]. Even studies of the erythropoietin (EPO) receptor are correct with the importance of dimerization for receptor activation, there EPO receptors by a single amino acid exchange, a cysteine residue introduces and leads to disulfide-linked homodimers, constitutively activated can be [Watowich, et al., Proc. Natl. Acad. Sci. USA 89: 2140-2144 (1992)].

In addition to homo- and heterodimerization of β-subunits as the critical step for receptor activation, a second important feature is that the formation of the final receptor complex by the CNTF family of cytokines occurs by a mechanism in which the ligand is successively in an ordered manner and binds to receptor components [Davis, et al., Science 260: 1805-1818 (1993); Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. Thus, CNTF first binds to CNTFRα, forming a complex which then binds gp130 to form an intermediate (referred to herein as αβ1 intermediate) that can not pass any signals since it has only a single β component before it finally reacted with LIFRβ to form a heterodimer of β-components, which then initiates signal transduction. Although a similar intermediate containing IL-6 bound to IL-6Rα and a single molecule gp130 was not directly isolated, we have assumed that it exists in analogy to its distant relative, CNTF, such as the fact that the final active IL-6 receptor complex includes two gp130 monomers. Overall, these findings led to a proposal for the structure of a receptor complex of the genus cytokines ( 1 ) in which each cytokine can have up to 3 receptor binding sites: a site that binds to a possible α-specificity component (α site), a site that binds to the first β signal-transducing component (β1 site), and a site which binds to the second β signal transducing component (β2 site) [Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. These 3 sites are occupied in a sequential manner, with the final step of complex formation - which results in dimerization of the β components - being crucial for the initiation of signal transduction [Davis, et al., Science 260: 1805-1818 (1993)].

If Cytokine binding induces receptor complex formation, activates the dimerization the β components the intracellular tyrosine kinase activity, which leads to the phosphorylation of a wide variety of substrates [lp, et al., Cell 69: 1121-1132 (1992)]. This activation of tyrosine kinase appears to be downstream for the following Events are crucial because inhibitors that inhibit tyrosine phosphorylation block, even later Events, such as gene induction, prevent [lp, et al., Cell 69: 1121-1132 (1992); Nakajima and Wall, Mol. Cell. Biol. 11: 1409-1418 (1991)]. Recently, we showed that a newly discovered family of non-receptor tyrosine kinases, which includes Jak1, Jak2 and Tyk2 (referred to as Jak / Tyk kinases) [Firmbach-Kraft, et al., Oncogene 5: 1329-1336 (1990); Wilks, et al., Mol. Cell. Biol. 11: 2057-2065 (1991)], and those with other cytokines play a role in signal transduction [, et al., Cell 74: 237-244 (1993); Silvennoinen, et al., Proc. Natl. Acad. Sci. USA (in press; 1993); Velazquez, et al., Cell 70: 313-322 (1992); Witthuhn, et al. Cell 74: 227-236 (1993)], with the cytoplasmic domains of the β subunits gp130 and LIFRβ in the absence Preassociate a ligand and tyrosine phosphorylated and activated after addition of Ligands [Stahl, et al., Science (submitted; 1993)]. Therefore, seem these kinases being the first step in intracellular signal transduction, those inside the cell as a result of ligand binding outside the cell is activated. Test systems for screening collections small molecules on specific agonist and antagonist activities The basis of this system will be described below.

Additionally have Brakenhoff et al (Cytokines, Vol. 3, No. 5, p. 496, abstract 279) two regions of the IL-6 molecule that are important for bioactivity are identified, neutralized by two groups (I and II) monoclonal antibody (mAb) are detected. Brakenhoff et al found that site I (recognized by group I mAbs) binding to the 80 kDa chain of the IL-6 receptor involved was while Group II on the interaction of the IL-6/80 kDa complex with the signal transduction chain gp130 is involved. Group II mAbs bind to the IL-6 residue Trp158 around. Random mutagenesis around Trp158 led to the isolation of a series mutants that bind to group I but not group II mAbs, which were biologically active as measured in a mouse B9 hybridoma assay. With two of these Mutants failed to induce IgG1 production when activated human CESS cells were tested, and one of these two mutants was able to detect the IgG1-inducing activity of wild-type IL-6 and also IL-6-induced fibrinogen and C1 inhibitor synthesis in human Hen G2 cells inhibit.

The CNTF family of cytokines plays an important role in a wide variety physiological processes, the possible therapeutic applications for both antagonists and agonists deliver.

SUMMARY OF THE INVENTION

• (a) die lösliche, die α-Spezifität bestimmende Komponente des Cytokinrezeptors; und
• (b) eine extrazelluläre Domäne einer β-Komponente des Cytokinrezeptors.

The invention provides a soluble cytokine antagonist protein capable of binding a cytokine to form a non-functional complex, wherein the cytokine activates a receptor by first binding an α-specificity determining component and then to β ₁ - and then binds to β ₂ signal transducing components, wherein the antagonist is heterodimeric and comprises:

• (a) the soluble α specificity component of the cytokine receptor; and
• (b) an extracellular domain of a β-component of the cytokine receptor.

Prefers the cytokine is IL-6 or CNTF.

Prefers is the extracellular domain (b) the extracellular domain from gp130.

More preferred is the cytokine CNTF, the α component (a) is sCNTFRα and the extracellular domain (b) is the extracellular domain from gp130; or the cytokine is IL-6, the α-component (a) is sIL-6Rα and the extracellular domain (b) is the extracellular domain from gp130.

The The invention also provides pharmaceutical compositions comprising a protein of the invention together with a pharmaceutically acceptable carrier.

The The invention also provides a protein of the invention, wherein the Cytokine IL-6 is the α component (a) sIL-6Rα is and the extracellular domain (b) the extracellular domain of gp130 is for use in a method of treatment one related to IL-6 Disease for use in the treatment of osteoporosis in one Patients with estrogen deficiency and for use in the treatment of multiple myeloma and cachexia.

One The subject of the present invention is the production of IL-6 antagonists which for the Treatment of IL-6 related diseases or disorders.

One Another object of the invention is the use of those described herein IL-6 antagonists for the treatment of osteoporosis.

One Another object of the invention is the use of those described herein IL-6 antagonists for the treatment of both the primary as well as the secondary Effects of cancer, including multiple myeloma.

Yet Another object of the invention is the use of the here described IL-6 antagonists for the treatment of cachexia.

BRIEF DESCRIPTION OF THE FIGURES

1 : Ordered binding of the receptor components in a model of a receptor of the genus cytokines. The model shows that cytokines contain up to 3 receptor binding sites and interact with their receptor components by first binding the possible α-component, followed by binding to β1, and then to β2. The β components of many cytokine receptors interact through membrane-adjacent regions (shaded fields) with the Jak / Tyk family of cytoplasmic protein tyrosine kinases. Signaling is initiated only after dimerization of the β components, as shown schematically by the tyrosine phosphorylations (P) of the β components and the Jak / Tyk kinases.

2 : CNTF inhibits IL-6 responses in a PC12 cell line (called PC12D) expressing IL6Rα, gp130, CNTFRα, but not LIFRβ. PC12D cells deprived of serum were incubated + IL-6 (50 ng / ml) in the presence or absence of CNTF as indicated. Some plates also received soluble IL6Rα (1 mg / mL) or soluble CNTFRα (1 mg / mL) as indicated. Cell lysates were subjected to immunoprecipitation with anti-gp130 and immunoblotted with anti-phosphotyrosine. Tyrosine phosphorylation of gp130 indicates IL-6-induced activation of the IL-6 receptor system that is blocked upon co-addition of CNTF.

3 : Scatchard analysis of iodinated CNTF binding on PC12D cells. PC12D cells were incubated with various concentrations of iodinated CNTF in the presence or absence of an excess of radioactive competitor to determine specific binding. The figure shows a Scatchard plot of the amount of iodinated CNTF that is specifically bound and gives data consistent with two binding sites with dissociation constants of 9 pM and 3.4 nM.

DETAILED DESCRIPTION THE INVENTION

The The invention described herein prefers the production of antagonists for each Cytokine, the an α specificity determining Component used, after binding of the cytokine to a first β-signal transducing component binds to form a non-functional intermediate, which then to a second β-signal transducing Component binds and so does β-receptor dimerization and subsequent Signal transduction causes. According to the invention, the soluble α-specificity determining Component of the receptor (sRα) and the extracellular domain the first β-signal transducing Component of the cytokine receptor (β1), where they heterodimers form (sRα: β1), which act as antagonists of the cytokine by releasing the cytokine bind to form a non-functional complex.

As described in Example 1, CNTF and IL-6 share the β1 receptor component gp130. The fact that CNTF forms an intermediate with CNTFRα and gp130 can be demonstrated in cells lacking LIFRβ (Example 1) in which the complex of CNTF and CNTFRα bind gp130 and the homodimerization of gp130 by IL-6 and IL- 6Rα prevents, which blocks the signal transduction. These studies provide the basis for the development of the IL-6 antagonists described herein since they show that when, in the presence of a ligand, a non-functional intermediate complex is formed can, consisting of the ligand, its α-receptor component and its β1-receptor component, this effectively blocks the action of the ligand. Other cytokines may use other β1 receptor components, which may also be used to produce antagonists according to the present invention.

So For example, in one embodiment of the invention, they are effective Antagonists of IL-6 or CNTF from heterodimers determine the extracellular domains of the α-specificity Components of their receptors (sIL-6Rα or sCNTFRα) and the extracellular domain from gp130. The resulting heterodimers, hereinafter referred to as sIL-6Rα: β1 respectively sCNTFRα: designated β1 act as high-affinity traps for IL-6 and CNTF, respectively, thus making the cytokine inaccessible make a signal-transducing complex with the native membrane-bound ones Forming forms of their receptors.

Even though soluble Ligand binding domains of the extracellular Part of receptors are somehow effective as traps for their ligands have proven to act as antagonists [Bargetzi, et al., Cancer Res. 53: 4010-4030 (1993); et al., Proc. Natl. Acad. Sci. USA 89: 8616-8620 (1992); Mohler, et al., J. Immunol. 151, 1548-1461 (1993); Narazaki, et al., Blood 82: 1120-1126 (1993)], the IL-6 and CNTF receptors unusual in this that the α-receptor components Ligand binding domains which, together with their ligands, are effectively soluble Act as receptor antagonists [Davis, et al., Science 259: 1736-1739 (1993); Taga, et al., Cell 58: 573-581 (1989)]. The according to the present Invention produced sRα: β1 heterodimers create effective traps for their ligands by using these ligands with affinities in the picomolar range (on the Basis of binding studies for CNTF to PC12D cells) bind without functional intermediates form.

The α and β ₁ receptor extracellular domains can be prepared by methods known to those skilled in the art. The CNTFRα receptor has been cloned, sequenced and expressed [Davis, et al., Science 253: 59-63 (1991), incorporated herein by reference in its entirety]. The cloning of LIFRβ and gp130 are described in Gearing et al. in EMBO J. 10: 2839-2848 (1991), Hibi, et al. Cell 63: 1149-1157 (1990) and in published PCT application WO 93/10151, published May 27, 1993, all of which are incorporated herein by reference in their entireties.

The Receptor molecules, the for execution useful in the present invention are, can by cloning and expression in a prokaryotic or eukaryotic Expression system are produced. The recombinant receptor gene can with the help of countless Methods are expressed and purified. The factor coding Gene can be subcloned into a bacterial expression vector, such as, but not limited to, pCP110.

The recombinant factors can to be cleaned with any technique that the subsequent formation of a stable, biologically active protein. For example, and without restriction, can the factors are recovered from cells either as soluble proteins or as inclusion bodies They are used quantitatively with 8M guanidine hydrochloride and Dialysis can be extracted. For further purification of the factors, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration.

The heterodimeric sRα: β ₁ receptors can be constructed using known fusion regions as described in published PCT application WO 93/1051, published May 27, 1993, entitled "Receptor of Oncostatin M and Leukemia Inhibitory Factor", which is entitled Preparation of β-receptor heterodimers, or they can be prepared by cross-linking the extracellular domains with chemical agents The domains used may consist of the entire extracellular domain of the α and β ₁ components, or they may consist of mutants or fragments thereof which maintain the ability to form a complex with its ligands and other components in the sRα: β complex.

In an embodiment According to the invention, the extracellular domains are constructed with the "leucine zipper" motif. For "leucine zipper" domains the human transcription factors c-jun and c-fos could be shown be that they are stable heterodimers in a 1: 1 stoichiometry form [bush and Sassone-Corsi, Trends Genetics 6: 36-40 (1990); Gentz, et al., Science 243: 1695-1699 (1989)]. Although the formation of jun-jun homodimers have also been shown, they are about 1000 times less stable than jun-fos heterodimers. Fos fos homodimers were not established.

The "leucine zipper" domain of either c-jun or c-fos is attached in frame to the C-terminal end of the soluble or extracellular domains of the above-mentioned receptor components by genetic engineering of chimeric genes. The fusions can be direct or they can have a flexible compound domain, such as the hinge region of human IgG or polypeptide compounds consisting of small amino acids such as glycine, serine, threonine or alanine, in various lengths and combinations. In addition, the chimeric proteins His-His-His-His-His (His ₆ ) [SEQ. ID NO. 1] to allow rapid purification by metal chelate chromatography, and / or epitopes for which antibodies are available to allow detection on Western blots, immunoprecipitation, or reduction / blocking of activity in bioassays.

In another embodiment becomes the sRα: β1 heterodimer by means of a similar one Method, but using the Fc domain of human IgG1 becomes [Aruffo, et al., Cell 67: 35-44 (1991)]. In contrast to the latter, must Formation of heterodimers biochemical, since chimeric molecules with the Fc domain as over Disulfide-linked homodimers are expressed. Consequently, homodimers can in conditions involving the interruption of disulfides between Favor chains, but have no effect on disulfides within the chain, reduced be. Then, monomers with different extracellular parts in equimolar Quantities are mixed and oxidized to a mixture of homo- and heterodimers to build. The components of this mixture are chromatographically Techniques separated. In another embodiment, the formation this type of heterodimers through genetic engineering and Expression of molecules, the from the soluble or extracellular Part of the receptor components followed by the Fc domain of hlgG, followed by the c-jun or c-fos "leucine zipper" motifs described above, affected [Kostelny, et al., J. Immunol. 148: 1547-1553 (1992)]. Since these "leucine zippers" predominantly form heterodimers, can they can be used to drive the formation of the heterodimers, where desired. As for the described chimeras Proteins using "leucine zipper" may be present these are also attached to metal chelates or an epitope. This attached domain can for the rapid purification by metal chelate chromatography and / or antibody to detection on Western blots, immunoprecipitation or reduction / blocking the activity to allow in biotests be used.

In another embodiment The invention provides the sRα: β1 heterodimers Expression as a chimeric molecules manufactured using flexible connection loops. One DNA construct that is the chimeric Protein encoded is designed to be two soluble or non-proteinaceous extracellular domains expressed in series by a flexible loop with each other are connected ("head to head ") Loop can completely artificially (e.g., polyglycine repeats, interrupted by serine or threonine at a certain distance) or she can of course occurring proteins (e.g., the hinge region from hlgG). molecules can be constructed, in which the sequence of the connected soluble and extracellular domains is swapped (e.g., sIL6Rα / loop / sgp130) or sgp130 / loop / sIL-6Rα) and / or, where the length is and the composition of the loop changes to the selection of molecules with desired ones To enable properties.

In another embodiment, the heterodimers prepared according to the present invention may be purified from cell lines co-transfected with the appropriate α and β ₁ components. Heterodimers can be separated from homodimers by methods available to those skilled in the art. For example, limited amounts of heterodimers can be recovered by passive elution from preparative non-denaturing polyacrylamide gels. In another embodiment, heterodimers can be purified by high pressure cation exchange chromatography. Excellent purification was achieved with a Mono S cation exchange column.

• 1) In der Osteoporose, die durch Erniedrigung der Östrogenniveaus in Post-Menopause-Frauen oder durch Ovariektomie verschlimmert werden kann, scheint IL-6 ein entscheidender Vermittler von Osteoklastogenese zu sein, die zur Knochenresorption führt [Horowitz, Science 260: 626–627 (1993); Jilka, et al., Science 257: 88–91 (1992)]. Entscheidenderweise scheint IL-6 nur eine größere Rolle im Zustand des Östrogenmangels zu spielen, und ist offensichtlich geringfügig an der normalen Knochenerhaltung beteiligt. Übereinstimmend damit deuten experimentelle Hinweise an, dass funktionsblockierende Antikörper gegen IL-6 die Anzahl an Osteoklasten verringern können [Jilka, et al., Science 257: 88–91 (1992)]. Während auch Östrogenersatztherapie angewendet wird, scheint es Nebeneffekte zu geben, die erhöhtes Risiko von endometrialem und Brustkrebs einschließen können. Folglich wären IL-6-Antagonisten wie hier beschrieben spezifischer, um die Osteoklastogenese auf ein normales Niveau zu reduzieren.
• 2) IL-6 scheint direkt mit multiplem Myelom etwas zu tun zu haben, indem es entweder in einer autokrinen oder parakrinen Art wirkt, um Tumorbildung zu fördern [van Oers, et al., Ann. Hematol. 66: 219–223 (1993)]. Darüberhinaus führen die erhöhten IL-6-Mengen zu unerwünschten sekundären Effekten wie Knochenresorption, Hypercalcämie und Kachexie; in wenigen Untersuchungen besitzen funktionsblockierende Antikörper gegen IL-6 oder IL-6Rα eine Wirkung [Klein, et al., Blood 78: 1198–1204 (1991); Suzuki, et al., Eur. J. Immunol. 22: 1989–1993 (1992)]. Daher wären IL-6-Antagonisten wie hier beschrieben vorteilhaft für sowohl die sekundären Effekte als auch für die Hemmung des Tumorwachstums.
• 3) IL-6 könnte ein Vermittler von Tumornekrosefaktor (TNF) sein, der zu Kachexie verbunden mit AIDS und Krebs führt [Strassmann, et al., J. Clin. Invest. 89: 1681–1684 (1992)], vielleicht durch Verringerung der Lipoprotein-Lipase-Aktivität in Fettgewebe [Greenberg, et al., Cancer Research 52: 4113–4116 (1992)]. Entsprechend wären die hier beschriebenen Antagonisten nützlich, Kachexie in solchen Patienten zu lindern oder vermindern.

The CNTF family antagonists described herein either bind or compete with the cytokines CNTF and IL-6. Accordingly, they are useful to treat diseases or disorders mediated by CNTF or IL-6. For example, therapeutic applications of IL-6 antagonists would include the following:

• 1) In osteoporosis, which may be exacerbated by lowering estrogen levels in post-menopausal women or by ovariectomy, IL-6 appears to be a critical mediator of osteoclastogenesis leading to bone resorption [Horowitz, Science 260: 626-627 ( 1993); Jilka, et al., Science 257: 88-91 (1992)]. Importantly, IL-6 appears to play only a major role in the condition of estrogen deficiency, and is apparently marginally involved in normal bone preservation. Consistent with this, experimental evidence suggests that functionally-blocking antibodies to IL-6 may reduce the number of osteoclasts [Jilka, et al., Science 257: 88-91 (1992)]. While estrogen replacement therapy is also being used, there appear to be side effects that increase the risk of endome trialem and breast cancer. Thus, IL-6 antagonists would be more specific as described herein to reduce osteoclastogenesis to normal levels.
• 2) IL-6 appears to be directly involved in multiple myeloma by acting either in an autocrine or paracrine manner to promote tumor formation [van Oers, et al., Ann. Hematol. 66: 219-223 (1993)]. Moreover, increased IL-6 levels lead to undesirable secondary effects such as bone resorption, hypercalcaemia and cachexia; in a few studies, functionally-blocking antibodies to IL-6 or IL-6Rα have an effect [Klein, et al., Blood 78: 1198-1204 (1991); Suzuki, et al., Eur. J. Immunol. 22: 1989-1993 (1992)]. Therefore, IL-6 antagonists as described herein would be beneficial for both the secondary effects and the inhibition of tumor growth.
• 3) IL-6 could be a tumor necrosis factor (TNF) mediator leading to cachexia associated with AIDS and cancer [Strassmann, et al., J. Clin. Invest. 89: 1681-1684 (1992)], perhaps by reducing lipoprotein lipase activity in adipose tissue [Greenberg, et al., Cancer Research 52: 4113-4116 (1992)]. Accordingly, the antagonists described herein would be useful in alleviating or reducing cachexia in such patients.

effective Cans, useful to these or other diseases related to the CNTF family or faults to treat be determined by methods known to a person skilled in the art [see, for Example, Fingl, et al., The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds. Macmillan Publishing Co., New York, SS. 1-46 (1975)]. Medicaments for use according to the invention contain the antagonists described above in a pharmacological acceptable liquid, solid or semi-solid carrier, tied to a carrier or a controlling molecule (e.g., antibodies, Hormone, growth factor, etc.) and / or in liposomes, microcapsules and controlled release devices (including antagonist-expressing Cells) integrated before administration in vivo. For example, that can Medicines one or more of the antagonists in an aqueous Solution, like sterile water, saline, Phosphate buffer or dextrose solution include. In another embodiment can the active agents in a solid (e.g., wax) or semi-solid (e.g., gelatinous) formulation contained in a patient can be implanted as needed for such treatment. Of the Routes of administration may be any type of administration known in the art be, including, but not limited to intravenously, intrathecal, subcutaneous, by injection into affected tissue, intra-arterially, intranasally, orally or through an implanted device.

The Administration may be in the distribution of the active agent of the invention through the body or result in a local area. For example, in some Conditions that affect distant areas of the nervous system, intravenous or intrathecal administration of the agent. In some situations For example, an implant containing active agent may be in or near the injured one Area are used. Suitable implants are closed but not limited to Gel foam, wax or microparticle-based implants.

EXAMPLE 1: CNTF COMPASSES WITH IL-6 UM THE BINDING AT GP130

MATERIAL AND METHODS

Material. A clone of PC12 cells responding to IL-6 (PC12D) was obtained from DNAX. Rat CNTF was prepared as described [Masiakowski, et al., J. Neurochem. 57: 1003-1012 (1991)]. IL-6 and sIL-6R were from R & D Systems bought. Antisera were raised in rabbits against a peptide derived from a region near the C-terminus of gp130 is (sequence: CGTEGQVERFETVGME) [SEQ.ID.NO.2], with the described Method [Stahl, et al., J. Biol. Chem. 268: 7628-7631 (1993)]. Antiphosphotyrosinmonoclonaler 4G10 was purchased from UBI and reagents for ECL from Amersham.

Signal transduction assays. Plates (10 cm) of PC12D were starved in serum-free medium (RPMI 1640 + glutamine) for 1 hour, then with IL-6 (50 ng / mL) + sIL-6R (1 μg / mL) in the presence or absence of rat CNTF at the indicated concentrations 5 Minutes at 37 ° C incubated. The samples were then subjected to anti-gp130 immunoprecipitation, SDS-PAGE and anti-phosphotyrosine immunoblotting as described [Stahl, et al., J. Biol Chem. 268: 7628-7631 (1993)].

RESULTS

The ability of CNTF to block IL-6 responses was measured with a PC12 cell line (called PC12D) expressing IL-6Rα, gp130, and CNTFRα, but not LIFRβ. As one would predict, these cells respond to IL-6, but not to CNTF ( 2 ), because LIFRβ is a required component for CNTF signal transduction is [Davis, et al., Science 260: 59-63 (1993)]. In agreement with results on other cell lines [lp, et al., Cell 69: 1121-1132 (1992)], PC12D cells give tyrosine phosphorylation of gp130 (as well as a variety of other proteins called CLIPs) in response to 2 nM IL-6 ( 2 ). Addition of recombinant soluble IL-6Rα (sIL-6Rα) increases the amount of gp130 tyrosine phosphorylation as reported in several other systems [Taga, et al., Cell 58: 573-581 (1989)]. Simultaneous addition of 2 nM CNTF with IL-6 greatly reduces tyrosine phosphorylation of gp130. Although a slight gp130 phosphorylation response remains in the presence of CNTF, IL-6 and sIL-6Rα, it is removed as the CNTF concentration is increased fourfold to 8 nM. Thus, in IL-6 responsive cells containing CNTFRα but not LIFRβ, CNTF is a fairly potent IL-6 antagonist.

EXAMPLE 2 Binding of CNTF to CNTFRα: β

MATERIAL AND METHODS

Scatchard analysis of CNTF binding. ¹²⁵ I-CNTF was prepared and purified as described [Stahl, et al., JBC 268: 7628-7631 (1993)]. Saturation binding studies were performed in PC12 cells at concentrations of ¹²⁵ I-CNTF ranging from 20 pM to 10 nM. The binding was performed directly on a monolayer of cells. The medium was removed from the wells and the cells were washed once with assay buffer consisting of phosphate buffered saline (PBS; pH 7.4), 0.1 mM bacitracin, 1 mM PMSF, 1 μg / ml leupeptin, and 1 mg / ml BSA. The cells were incubated in ¹²⁵ I-CNTF for 2 hours at room temperature, followed by 2 quick washes with assay buffer. The cells were lysed with PBS containing 1% SDS and counted in a Packard gamma counter at 90-95% efficiency. Non-specific binding was defined by the presence of a 100-fold excess of unlabeled CNTF. Specific binding ranged from 70% to 95%.

RESULTS

The equilibrium constant for the binding of CNTF to CNTFRα: β1 was estimated by Scatchard analysis of iodinated CNTF binding on PC12D cells ( 3 ). The data are consistent with a two-binding-site form that has dissociation constants of 9 pM and 3.4 nM. The low affinity binding site corresponds to the interaction of CNTF with CNTFRα, which has a Kd close to 3 nM [Panayotatos, et al., J. Biol. Chem. 268: 19000-19003 (1993)]. We interpret the high affinity complex as the intermediate containing CNTF, CNTFRα and gp130. An Ewing sarcoma cell line containing CNTFRα, gp130 and LIFRβ and thus giving stable tyrosine phosphorylation in response to CNTF shows a very similar two-binding-site form with dissociation constants of 1 nM and 10 pM (Wong, et al., Unpublished data). , Thus, it is apparent that equally high affinity CNTF binds to a complex containing only CNTFRα and gp130, as it binds to a complex additionally containing LIFRβ, thus providing the feasibility of creating the sRα: β described herein ₁ antagonist is shown.

SEQUENCE LISTING

Claims (11)

A soluble cytokine antagonist protein capable of binding a cytokine to form a non-functional complex, wherein the cytokine activates a receptor by first binding an α-specificity-determining moiety and subsequently to β ₁ - and then β ₂ - Signal transducing components bind, wherein the antagonist is heterodimeric and comprises: (a) the soluble, α-specificity determining component of the cytokine receptor; and (b) an extracellular domain of a β ₁ component of the cytokine receptor. The protein of claim 1, wherein the cytokine is interleukin-6 (IL-6) or ciliary neurotrophic factor (CNTF). A protein according to claim 1 or 2, wherein the extracellular domain (b) is the extracellular domain from gp130 is. Protein according to claim 2 or 3, wherein the cytokine CNTF and component (a) sCNFTRα and the extracellular domain (b) the extracellular domain from gp130 is. Protein according to claim 2 or 3, wherein the cytokine IL-6, the α component (a) sIL-6Rα and the extracellular domain (b) the extracellular domain from gp130 is. A DNA sequence comprising a protein according to any one of claims 1 to 5 coded. Medicament comprising a protein according to one of claims 1 to 5 together with a pharmaceutically acceptable carrier. A protein according to claim 5 for use in a method for the treatment of IL-6 related disease or disorder. A protein according to claim 5 for use in a method of treating osteoporosis in one Patients with estrogen deficiency. A protein according to claim 5 for use in a Method for the treatment of multiple myeloma in a patient. A protein according to claim 5 for use in a Method of treating cachexia in a patient.